1. Field of the Invention
The present invention relates generally to the field of biochemistry and molecular biology. More particularly, it concerns methods to isolate and identify biologically active polypeptides.
2. Description of Related Art
Many of the most effective modern therapeutics are polypeptide molecules such as monoclonal antibodies. In the case of antibodies, the mammalian immune system provides a highly adapted system for development of antibody molecules that are specific for a given therapeutic target. Modern molecular biology techniques allow the sequences for these antibodies to be isolated, such that therapeutics based on the antibody sequences can be mass produced in fermentation systems. Unfortunately, the development of antibody therapeutics is limited in that the therapeutic target must be known, be antigenic and be accessible on the surface of a target cell.
Accordingly, methods for identifying candidate biologically active polypeptides by using molecular libraries are being explored. However, any such system requires that the library have sufficient diversity to interrogate a vast range of candidate molecules. Moreover, any assay using such a library must provide a system for determining the coding sequence for polypeptides that are identified in a binding or biological activity screen. In some cases, the polypeptide sequence can be directly determined, such as by mass spectroscopy, but such a method requires a large amount of each given polypeptide. Alternatively, the polypeptide can be tethered to its nucleic acid coding sequence by some method. Such methods based on tethering are generally referred to as biological display (e.g., phage display).
Phage display technology has been successful as providing a vehicle that allows for the selection of a displayed protein by providing an essential link between nucleic acid and the activity of the encoded polypeptide (for a review see, e.g., Clackson and Wells, 1994). In this case, filamentous phage particles act as genetic display and packages proteins on the outside of the particle and the genetic elements that encode them on the inside. However, phage display relies upon the creation of nucleic acid libraries in vivo in bacteria and this places a limitation on library size that can be used. Additionally, all potentially useful candidate polypeptides are fused to phage sequences for display and such fusion may interfere with the ability of the polypeptide function. Thus, there remains no efficient system for screening and identification of biologically active polypeptide molecules.